In this proposal we describe the development of a novel genetic manipulation system termed "genomic engineering" that will permit efficient, unlimited and directed modification of a chosen genomic locus in Drosophila. Genomic engineering is a two-step process. First, through an optimized gene targeting routine, a founder knock-out line is generated by deleting the target gene and replacing it with a small DNA recombination site of phage integrase FC31. Second, DNA integration by FC31 is used to reintroduce modified target gene DNA into the native locus in the founder knock-out line. Since FC31 mediated DNA integration is highly efficient and non-discriminating, virtually any desired genetic modification can be generated with high throughput efficiency. Genomic engineering will have a profound and revolutionary impact as a tool for directed engineering of the Drosophila genome. First, genomic engineering overcomes the inherent inefficiency and limitations of homologous recombination. It provides a virtually unlimited approach to generate any desired mutant allele of a target gene for genetic, biochemical and cell biologic assays. Second, for constructing useful and informative human disease models, genomic engineering makes it possible to precisely replicate the human genetic lesions into the Drosophila homologous genes. Finally, by systematically generating founder knock-out lines for conserved and essential genes in the long term, we will transform Drosophila into a far more efficient and versatile genetic model organism that will also be vastly more accessible and attractive to non-fly researchers for addressing their biological questions. PUBLIC HEALTH RELEVANCE: Drosophila is a leading genetic model system for addressing crucial biological questions in human diseases. Genomic engineering is a new powerful genetic tool that will significantly facilitate the analysis of complex disease pathways and the construction of better disease models in Drosophila.